PROJECT SUMMARY/ABSTRACT Many bacterial species in the phylum Firmicutes produce spores. The spores of commensal and pathogenic bacteria play a crucial role in host entry and the initial encounter with the immune system. How bacterial spores interact with host immunity, however, remains poorly understood. In the proposed research project, we investigate spore-immune interactions and their impact on inflammation and tissue homeostasis in the intestinal mucosa. We discovered that RNA-sensing toll-like receptors (TLRs)?TLR7 and TLR13 in mice and their human counterparts?played a crucial role in detecting and triggering immune responses to the spores of Bacillus anthracis (BA), the etiologic agent of anthrax. BA spores harbored high amounts of RNA whose TLR- stimulating activity was retained after extraction and purification. This RNA was mainly located in the outermost layer of the spore, or the exosporium. TLR7/13 sensing of BA spores led to type I interferon (IFN-I) production from host cells. These findings prompted us to search commensal bacterial spores for similar structural features and functional properties. For this investigation, we devised a novel protocol that enabled spore isolation from fecal samples or intestinal luminal contents without ex vivo culture. This protocol allowed us to detect gut microbiota-derived spores with high RNA content and IFN-I-inducing capacity. These findings, in conjunction with the emerging evidence that tonic IFN-I signaling protects against colitis, colon cancer, and enteric viral infection, led us to formulate the central hypothesis that TLR sensing of spore-associated RNA is a pivotal mechanism by which commensal bacterial spores induce IFN-I-dependent tissue-protective responses in the intestinal mucosa. In the proposed research, we will verify the central hypothesis by pursuing the following specific aims: to identify the commensal bacterial species in the mouse and human gut whose spores possess an RNA-laden exosporium and IFN-I-inducing activity (Aim #1); and to establish the role of commensal bacterial spores and spore-sensing TLRs in inducing IFN-I production and suppressing inflammatory damage in the intestinal mucosa (Aim #2). Our study will advance our understanding of mucosal physiology and pathology by identifying commensal bacterial spores as major inducers of intestinal IFN-I production and demonstrating their role in immune regulation and tissue protection.